Serotonin (5-HT) transporters (SERTs) are critical determinants of synaptic 5HT availability in the CMS and periphery and major targets for antidepressants and psychostimulants including cocaine and MDMA. Endogenous mechanisms that modulate SERT activity are emerging, but as yet remain ill defined. Past studies have identified SERTs as phosphoproteins, regulated acutely (minutes) by multiple signaling pathways that influence transporter trafficking and intrinsic activity. In the current proposal, we continue our investigations of SERT regulation, bridging heterologous model systems with studies using in vitro and in vivo studies of native preparations. In Specific Aim I we evaluate the role of protein kinase G (PKG)-linked signaling pathways in modulation of SERT trafficking and SERT protein/protein interactions as downstream consequences of GPCR activation in vitro and in vivo. Preliminary studies here reveal PKG-dependent changes in SERT surface density, 5-HT uptake, SERT currents and 5-HT clearance in vivo and support a direct role for SERT phosphorylation in GPCR/PKG linked modulation. In Specific Aim II, we examine the role of p38 mitogen associated protein kinase (MAPK) in modulating SERT intrinsic activities. Preliminary studies here provide evidence for catalytic modulation of SERT targeting surface transporters, dependent on PP2A function for p38 MAPK regulation, and the ability of p38 MAPK to directly phosphorylate SERT. In specific aim III, we investigate the degree to which basal and PKC/PKG/p38 MAPK-mediated alterations in SERT activity are evident in human SERT coding polymorphisms. Preliminary findings on this Aim reveal the existence of multiple SERT alleles that display altered kinase-mediated regulation, alleles that may be enriched in populations at risk for 5-HT linked disorders and which offer novel opportunities for insights into molecular mechanisms of SERT regulation/therapeutics.